The present invention concerns the field of development and quality control of devices and materials for fluid handling, control, flow, and deposition.
At presen methods for analyses in these applications typically rely upon detection of fluorescent or chromogenic dyes using a time-lapse or high-speed video device. For flow analysis, a fine stream of the fluorescent or chromogenic detection agent can be introduced into the system. However, information obtained using these methods is of limited utility with respect to a significant area of the flow channel because of limited sensitivity and rapid diffusion and dilution of the detected agent.
Resonance light scattering (RLS) particles have been shown to provide highly sensitive labels in bioanalytical assays in a variety of different formats. Such uses are described, for example, in Yguerabide at al., PCT/US97/06584, Yguerabide et al., PCT/US98/23160, Yguerabide et al, U.S. application Ser. No. 08/844,217, now U.S. Pat. No. 6,214,560, and Yguerabide et al., U.S. application Ser. No. 08/953,713, and the patents and publications cited in the backgrounds thereof, all of which are hereby incorporated by reference herein in their entireties, including drawings.
Particles with similar composition have also been used in connection with electron microscopy and as cytological stains, utilizing their associated absorbence properties.
The present invention relates to the use of resonance light scattering (RLS) particles as tools to guide and refine design, manufacture, and quality control parameters in production of fluid-containing devices and processes. The use of such particles is particularly advantageous for small volume devices, such as micro, nano, and pico volume fluidic, capillary, or deposition instruments, devices, and products.
These applications of RLS particles relate to the capability provided by such particles to obtain detailed information associated with micro-scale, nano-scale, and pico-scale flow, as well as static properties or parameters, using appropriately formulated RLS particles. Such information can be readily obtained using simple instrumentation for detection, or even using detection by eye with appropriate magnification, though more complex apparatus, especially those having electronic analysis and/or control capabilities are advantageous in many applications.
Using such RLS particles, it is possible to detect light scattering from the particles continuously, without the bleaching experienced with fluorophores, and with very high sensitivity and signal stability. Detection can even be readily performed on single particles.
Thus, in a first aspect, the invention provides a method for determination of a dynamic property of a fluid volume. The dynamic property is determined by determining the distribution or location or both of at least one light scattering particle using detection of light scattered from the particle or particles in at least a portion of said fluid volume. For some devices it may be useful to view the particles in the entire device simultaneously, while in other cases, it may be useful to view only a portion of the device, e.g., a valve, flow channel, or mixing chamber. In still other cases, it may be advantageous to follow a particle or set of particles as they are transported through a device or portion of a device.
A variety of flow properties can be involved, and the determination can involve one or more than one such property, determined simultaneously or sequentially. For example, the dynamic property may be flow rate, or particle distribution. In certain embodiments, the particles are directly or indirectly attached to a biological molecule such as a nucleic acid molecule such as nucleic acid probe, a polypeptide such as an antibody or antibody fragment, a lectin, a carbohydrate, or a cell. Thus, the attached light scattering particle provides a method for determining the distribution of biological molecule or cell in a volume or on a surface. In preferred embodiments, the device is an array, or other device on which molecules are deposited or bound in a localized manner, e.g., spotted on a solid phase surface or deposited in a well.
Similarly, in preferred embodiments, the dynamic property is uniformity (or lack of uniformity) of drying on a solid surface. This is particularly applicable to development and quality control of arrays, e.g., nucleic acid or polypeptide arrays. Uniformity (or lack of uniformity) can be uniformity across discrete areas, where the areas are evaluated individually (though the individual evaluations can then be compared), thereby providing a larger scale evaluation or comparison). Examples of such discrete areas are the individual features on a planar array. Alternatively, the uniformity can be evaluated across multiple discrete areas. As an example, the number of functional probes bound in the various features on a planar array can be evaluated. In either type of evaluation, lack of uniformity is typically shown by a pattern to the particle distribution. Such a pattern may, for example, be irregular differences in concentration or density of RLS particle, or a gradient in such concentration or density across an area or volume. In such embodiments, the uniformity can be evaluated continuously, or at discrete times, or at endpoint, or in combinations.
In preferred embodiment, the deposited volume and/or number of features is as described for embodiments of other aspects involving arrays herein.
In another example, the dynamic property is a flow pattern in a device or portion of a device. For example, the device may be a multi-channel device. Such a flow pattern, can for example, be the distribution of particles across a flow channel, presence and/or size of eddies or turbulence zones, flow velocity in a portion of a device, or a flow velocity profile across a channel, chamber, or other device portion.
In yet another example, the dynamic property is fluid mixing. The fluid mixing may be evaluated in one or more portions or elements of a device, or throughout the entire device. For example, the portion may, for example, be a mixing chamber, a port, a flow channel, a pump, or a flow channel intersection or junction. Such fluid mixing (as well as flow patterns and other properties or parameters) can be evaluated as a function of device parameters and/or other process parameters. Such other process parameters include, for example, fluid type (e.g., identity of solvent), electrical conductivity of the fluid, presence or absence or amount of one or more dissolved or suspended species, and viscosity of the fluid.
In preferred embodiments, the device is a small volume device, which may, for example, be a micro volume device such as a microchannel device; a nano volume device; a pico volume device; an array chip, plate, or slide; a pump, a port; a valve; a spotting pin; a channel junction, or a jet head.
Device and process evaluations can also be performed in devices or portions of multiple devices that have fluid connection. In this way, interactions between devices in a system can be determined and adjusted in beneficial ways.
In another aspect, the invention provides a method for analyzing deposition characteristics of features (spots) on an array, or other surface arrangement. The method involves depositing at least one fluid volume on a portion of a solid substrate, where the fluid volume contains a plurality of light scattering particles, and detecting the light scattering particles by detecting light scattered from the particles. The distribution or number or both of the particles is indicative of the deposition characteristics. Alternatively, another molecule(s) can be deposited on the array or other surfaces to which light scattering particles can be directly or indirectly bound. Then the presence, amount, and/or distribution of the light scattering particles serves as an indicator of the presence, amount, and/or distribution of the deposited molecule(s). The determinations can also involve other deposition characteristics.
Preferably the array contains at least 5, 10, 50, 100, 200, 500, 1000, 5000, 10000 spots, or even more. In particular embodiments, the number of features on the array is within a range between any two of the feature numbers just provided, inclusive of the endpoints.
Various deposition characteristics of array spots or other spots on a solid phase may be evaluated using the present method. For example, the deposition characteristic can be uniformity (or non-uniformity) of deposition. The uniformity can, for example, be evaluated as a 2-dimensional distribution of particles within a spot or spots, or as a deposition volume, and/or the uniformity of particle number in deposited spots. The distribution of particles can, for example, be used as an indicator of the distribution of probe molecules in a deposited spot or spots. Likewise, the deposition characteristic can be a drying pattern, for example the distribution of probe molecules during drying of the spot or spots, or a distribution of an active moiety during and/or after post-spotting processing.
The determination of number and/or distribution of a molecule or other detectable item can involve assay of number and/or distribution of functional components. For example, target, probe, or other binding molecule (e.g., an antibody or oligonucleotide probe) can be deposited on a surface. Then a binding assay can be performed, utilizing light scattering particles as described, to determine the number and/or distribution of probe (or other) molecules that have a particular binding function (e.g., specific binding and/or binding strength). Such assays can likewise be used to determine binding kinetics, e.g., by monitoring binding on a surface over time and/or binding stability (e.g., by using conditions of a particular stringency, or a range of stringencies). Monitoring over time can, for example, be performed using time lapse or video techniques. In preferred embodiments, the deposition characteristic determined is, or is indicative of, functional binding, for example, in nucleic acid hybridization, protein-protein interaction, and ligand-receptor binding.
As used herein, the term xe2x80x9carrayxe2x80x9d refers to a device having a solid phase surface which has a plurality of features in distinct, physical locations, typically separated by blank or empty areas. The xe2x80x9cfeaturesxe2x80x9d are locations where particular molecules, often biomolecules, are immobilized for conducting assays. In most current arrays, the immobilized molecules are probes that bind to target molecules in a sample or samples applied to the array. As used herein, the term xe2x80x9carrayxe2x80x9d is used as a general term for any such device. As used herein, the term xe2x80x9carray chipxe2x80x9d refers to an array with a planar solid substrate with surface area of 1 in2 or less; the term xe2x80x9carray slidexe2x80x9d refers to an array with a planar solid substrate with a surface area greater than 1 in2 up to 4 in2 inclusive; the term xe2x80x9carray platexe2x80x9d refers to an array with a solid substrate with a generally planar surface. In some embodiments, the plate has depressions, e.g., wells, for containing liquids.
In connection with arrays, the terms xe2x80x9cfeaturesxe2x80x9d and xe2x80x9cspotsxe2x80x9d are used synonymously. The features may, for example, be particular areas on a flat surface, wells, or channels (which may be oriented in a flat, viewing plane of the array, or end-on to a flat, viewing plane of the array). Preferably the features are particular locations on a flat surface, e.g., specific oligonucleotide or polypeptide-containing areas on a glass or plastic slide or chip.
In particular embodiments, the small volume deposited at one or more features is 1 pL to 100 nL, preferably 10 pL to 10 nL, more preferably 50 pL to 10 nL, still more preferably 50 pL to 1 nL. In additional particular embodiments, the small volume deposited is 50 pL to 500 pL, 50 pL to 200 pL, 10 pL to 200 pL, 10 nL to 200 nL, or 200 nL to 2 xcexcL. Also in particular embodiments, the volume deposited is in a volume range described by taking any two different particular volumes just specified as the inclusive endpoints of the range.
As in the previous aspect, in preferred embodiments the array has at least 5, 10, 50, 100, 200, 500, 1000, 5000, 10,000 features, or even more, or the number of features is in a range described by taking any 2 different values, as described, as the endpoints of the range.
The invention also provides a method for analyzing fluid flow in at least a portion of a device, preferably in a small volume device, more preferably in a plurality of portions of a small volume device. The method involves inserting a suspension of light scattering particles in the device, illuminating the light scattering particles in a plurality of portions of the device, and detecting the presence of light scattering particles as an indication of the fluid flow. The flow can, for example, be continuous, stopped, or pulsatile flow.
The portion or device may be any that is appropriate for fluid flow that allows, or can be adapted to allow illumination of particles within or on the device or portion of interest. Thus, preferably the device has at least a portion that is exposed or transparent to light, preferably to visible wavelength light, preferably the incident light beam is provided by laser or collimated incident light beam.
In connection with flow devices and/or other fluid-containing devices, light scattering particles can also be used to monitor binding of molecules in solution to the interior walls of the device. In many cases, such binding is undesirable, for example, as it results in loss of a significant portion of sample or because it makes quantitation of material or process results problematic or questionable. In preferred embodiments, determination of binding can be carried out similarly to binding determinations on an exposed surface (e.g., on a microarray). For example, in an exemplary embodiment, the solution containing the molecule of interest is placed in or flowed through the device or portion of a device. Unbound material is washed out of the device or portion, and bound molecules are detected by directly or indirectly binding light scattering particles to the bound molecules and detecting the light scattering particles as an indication of the presence of the bound molecules.
Flow detection and analysis can be performed in various ways depending on the flow property or properties of interest. For example, time lapse imaging can be used to provide particle images at discrete time points, extended exposures can be used to provide trace lines showing particle paths, and video images can be used to provide moving particle images. Combinations and other options can also be used.
In addition, in another aspect, the invention provides a method for analyzing at least one surface characteristic of a solid substrate or porous matrix. The method involve detecting the distribution or number or both of the light scattering particles on at least a portion of the substrate, e.g., a surface, by detecting light scattered from the particles, following treatment of at least a portion of the substrate with at least one fluid volume containing a plurality of light scattering particles. The distribution or number or both of the particles is indicative of the characteristic.
As used herein in connection with suspensions of light scattering particles, the terms xe2x80x9ctreatmentxe2x80x9d, treatingxe2x80x9d and words of like import refer to contacting a material or composition with the suspension, and can also include additional processes, for example, non-covalent binding, covalent binding, and washing.
In the context of substrate analysis, e.g., surface analysis, the terms xe2x80x9ccharacteristicxe2x80x9d, xe2x80x9csurface characteristicxe2x80x9d, and xe2x80x9cmatrix characteristicxe2x80x9d refer to a physical, chemical, and/or electrical property of the solid substrate, e.g., texture, planarity, porosity, surface charge, surface charge uniformity, hydrophobicity, hydrophilicity, reactivity, and combinations thereof, as well as other properties. One of ordinary skill in the art will recognize that the characteristics that can be analyzed are those that affect the number and/or distribution of particles on the surface, either directly or indirectly (e.g., through another component that is attached or becomes attached, directly or indirectly, to the particles).
Thus, in preferred embodiments, the characteristics analyzed include one or more of surface on matrix uniformity, uniformity of one or more coatings, uniformity of charge, uniformity of hydrophilicity, uniformity of hydrophobicity, and uniformity of charge density. The characteristic can concern a surface or surfaces and/or substrate through at least a portion of a porous matrix.
The terms xe2x80x9chydrophobicityxe2x80x9d and xe2x80x9chydrophilicityxe2x80x9d have their usual technical meaning, referring to whether a material does not associate readily with water, or associates readily with water, respectively.
Also in preferred embodiments, the solid substrate is a glass substrate, a functionalized glass substrate, a plastic substrate, a silicon substrate, a membrane substrate, a metallic substrate, or combinations thereof.
The determination of light scattering particles on the surface can be performed with illumination and detection on the same side of the substrate, e.g., membrane (for either transparent or non-transparent membranes or other substrates) or from opposite sides (for essentially transparent membranes or other substrates, and substrates that can be made essentially transparent).
In the context of this invention, xe2x80x9cmembranexe2x80x9d refers to a thin, flexible impermeable or microporous material, preferably synthetic material. Preferably pores or channels in the membrane are no larger than 20 xcexcm, more preferably no larger than 10, 5, 2, 1, 0.5, 0.2 or 0.1 xcexcm, or in a range specified by any two of these specified endpoints. Preferably, a membrane is a uniform sheet of material with essentially uniform composition, e.g., a film, though in some embodiments a membrane is fibrous material, e.g., woven or matted fibrous material. Examples of commonly used materials include nylon, nitrocellulose, polyvinylidene fluoride (PVDF), and cellulose.
As used herein, the term xe2x80x9cmatrixxe2x80x9d or xe2x80x9cmatrix materialxe2x80x9d refers to a porous material, preferably a microporous material. A porous material is one with channels that allow entry or passage of fluid and/or air. Such channels may be discrete or interconnecting, and may be through channels and/or blind channels, but are preferably through channels. Preferably such passages are of sufficient size to allow entry or passage of light scattering particles of at least 1 nm diameter, more preferably at least 10, 20, 30, 40, 60, 80, 100, 120, or 150 nm. Preferably the channels are, on average, at least 50, 100, 200, 400, 600, 800, or 1000 nm in cross-section. The degree of porosity can vary, e.g., representing at least 1, 2, 5, 10, 20, 40, or 50% or even more of a surface of a material. Thus, matrix materials include such exemplary materials and items as membrane filters, fibrous filters, and sintered glass filters.
The term xe2x80x9cfunctionalizedxe2x80x9d refers to a chemical modification that prepares a material, e.g., a glass, plastic or metal surface for subsequent chemical interaction by attaching or creating suitable functional groups or moieties. Thus, for example, an analysis can determine the number and/or density of accessible groups on a surface after functionalization.
As used herein, the term xe2x80x9cfluidxe2x80x9d refers to a material or combination of materials that is liquid under the relevant pressure and temperature conditions, e.g., at room temperature and one atmosphere.
In the context of this invention, the term xe2x80x9cdevicexe2x80x9d means an article of manufacture that includes one or more channels or reservoirs or locations for fluid to be present, e.g., for flow or deposition.
The term xe2x80x9cmicrochannelxe2x80x9d refers to a channel of sufficient size to allow fluid flow, preferably a channel generally in the form of a tube, that has mean cross-sectional measurement of 3 mm or less. In particular, embodiments the channel is 2 mm or 1 mm or less, or 0.5 mm or less, or 100 xcexcm or less, preferably 10 xcexcm or less or 100 xcexcm or less, still more preferably 80 nm or less, or 60 nm or less, or 40 nm or less, or 20 nm or less.
xe2x80x9cMicroscalexe2x80x9d refers to devices or portions of devices or processes with dimensions of 3 mm or less, preferably 1000 xcexcm or less, generally in the range of 1-500 xcexcm, for functional parts or processes. Thus, channels, junctions and the like are typically of such dimensions.
xe2x80x9cNanoscalexe2x80x9d refers to devices or portions of devices or processes with dimensions of 1000 nm or less, generally in the range of 1-500 nm, for functional parts or processes. Thus, channels, junctions, and the like are typically of such dimensions.
xe2x80x9cMicrofluid dynamicsxe2x80x9d refers to the fluid dynamics in microscale systems, preferably in systems with flow channel dimensions of 1 mm or less, 500 xcexcm or less, 100 xcexcm or less, 500 nm or less, 100 nm or less, 50 nm or less, or even smaller. Thus the term refers to the fluid behavior in such channels.
The term xe2x80x9csubfluidic regionxe2x80x9d refers to a portion of a flow channel or reservoir. Generally the term is used in connection with the behavior of fluids in such a sub-region in connection with microscale or nanoscale processes or channels or reservoirs. Likewise, the term xe2x80x9csub-flow patternxe2x80x9d refers to the fluid flow pattern or behavior in a sub-region of a flow channel or reservoir, generally microscale or nanoscale. Such regions and flows can be monitored using the methods of the present invention.
xe2x80x9cMicrofabricationxe2x80x9d refers to the techniques and processes of producing a microscale or nanoscale device. Exemplary techniques include photoetching, laser shaping, micromachining, and the like. The method utilized will depend on factors such as the scale and the materials being utilized.
The term xe2x80x9cfluid depositionxe2x80x9d refers to the process of placing a volume, generally a small volume, on a solid phase surface. The deposition may, for example, be on a flat surface, or in a depression, or cavity in the surface, or on the walls of a tube through a solid phase material. Exemplary methods include those commonly used for producing microarrays. A variety of such deposition methods, as well as other factors in production of arrays, are described in Microarray Biochip Technology, Mark Schena, ed., Eaton Publishing, Nattick, Mass., 2000, and are applicable for the present methods. Deposition methods include, for example, piezoelectric, inkjet, solenoid/piston, and pin spotting. Deposited volumes are preferably 1 xcexcL or less, 0.5 xcexcL or less, 0.1 xcexcL or less, or more preferably 10 nL (nanoliters) or 1 nL or less, or even 500 pL (picoliters) or less, 200 pL or less, 100 pL or less, or 50 pL or less.
The term xe2x80x9csmall volumexe2x80x9d as used herein refers to a volume of 10 mL or less, preferably 5 mL or less or 1, 0.5, 0.1 mL or less, more preferably 10 xcexcL or less, or 1, 0.5, 0.1 xcexcL or less, still more preferably 10 nL, 1 nL, 0.5 nL, or 0.1 nL or less. Still smaller volumes are also included. Thus, a small-volume device has a volume within the device within the limits described. The term may also be used in connection with portions of a device, or a process, or sub-process.
xe2x80x9cMicrovolumexe2x80x9d refers to volumes of equal to or less than 1000 xcexcl, generally in the range of 1 to 1000 xcexcl; in exemplary embodiments 100-1000 xcexcl, 200-800 xcexcl, 100-500 xcexcl, 1-500 xcexcl, 800 xcexcl or less, 500 xcexcl or less, or 200 xcexcl or less.
xe2x80x9cNanovolumexe2x80x9d refers to volumes of 1000 nL or less, generally in the range of 1 to 1000 nL, in exemplary embodiments 1-600 nL, 1-400 nL, 100-1000 nL, 100-600 nL, 800 nL or less, 500 nL or less, or 200 nL or less.
xe2x80x9cPicovolumexe2x80x9d refers to volumes of 1000 pL or less, generally in the range of 1 to 1000 pL, in exemplary embodiments, 1-600 pL, 1-400 pL, 100-1000 pL, 100-600 pL, 800 pL or less, 500 pL or less, or 200 pL or less.
xe2x80x9cResonance light scattering particlesxe2x80x9d (RLS particles) refers to particles that elastically scatter incident light with high efficiency. Preferably the particles are metal or metal-like particles. Preferred examples include gold particles, silver particles, and mixed composition gold and silver particles as well as particles containing at least 1, 5, 10, 25, 50, or 5% by weight of gold or silver or a combination of gold and silver. Examples of mixed composition particles are particles with silver surrounding a gold core, and gold over silver. For silver particles, a thin outer layer of gold can be advantageous, e.g. by providing light scattering characteristics of a silver particle while stabilizing the particle with the gold outer layer. Particles are also included that contain or are composed of other materials that have sufficient light scattering intensity to allow use as labels or as flow markers, preferably with particles sizes of 1-500 nm.
The size of RLS particles can be selected as needed or useful for particular applications. For example, particles can be selected to provide different colors on scattering of white, or more generally polychromatic light. Likewise, it may be beneficial to select particles of a particular sizes or with certain size limitations based on the dimensions of the fluid-containing portions of the device. In many cases, it is preferable to utilize particles that are small compared to the dimensions of the fluid channel or chamber, e.g., xe2x89xa6⅕, {fraction (1/10)}, or {fraction (1/20)}th. In many applications such size limitations are useful so that that particles will move freely through the device, and preferably will respond to eddies, turbulence, and other sub-features of the flow in the device to allow determination of those characteristics.
By xe2x80x9cmetal-likexe2x80x9d particles is meant any particle or particle-like substance that is composed of metal, metal compounds, metal oxides, semiconductor (SC), superconductor, or a particle that is composed of a mixed composition containing at least 0.1% by weight of metal, metal compound, metal oxide, semiconductor, or superconductor material.
By xe2x80x9ccoatedxe2x80x9d particle is meant a particle has on its surface a layer of additional material. The layer is there to chemically stabilize the particle in different environments, and/or to bind specific molecules by molecular recognition means. Such coatings include, for example, inorganic and organic compounds, polymers, proteins, peptides, hormones, antibodies, nucleic acids, receptors, and the like. As described in the Yguerabide references cited herein, coated metal-like particles have similar light scattering properties as compared to uncoated metal-like particles, both of which have superior light scattering properties as compared to non-metal-like particles.
By xe2x80x9cnon-metal-likexe2x80x9d particles is meant particles that are not composed of metal, metal compounds, superconductor, metal oxides, semiconductor, or mixed compositions that are not composed of at least 0.1% by weight of metal, metal compound, metal oxide, superconductor, or semiconductor material.
In this invention, it may be advantageous to provide a plurality of different particles, where each of the plurality is separately distinguishable. The plurality of different particles means that there is one or more individual particles, generally a large number of individual particles, of each of the different, separately distinguishable particles. The composition, size, and shape of the particles are chosen to provide distinguishable light scattering particles, e.g., different colors and/or different intensities. For example, roughly spherical gold particles of 40, 60, and 80 nm diameter can be used to provide distinguishable colors when illuminated with white (or polychromatic) light. In particular embodiments, 2, 3, 4, 5, 6, or even more distinguishable particles are used. The plurality of different particles can, for example, be used to analyze mixing of fluids from two different sources, e.g., from two different channels within a device, or to visualize the mixing of a small volume as it is combined with a larger volume. Such different particles can be provided, for example, using gold particles of different sizes, and/or silver particles of suitable size to provide distinguishable colors (e.g., two or more of 40, 60, and 80 nm gold particles, and 40 nm silver particles. Those skilled in the art will recognize a variety of other applications for multiple distinguishable particles.
Also, non-spherical particles may be used to provide useful information on flow or fluid properties, e.g., flow rate, viscosity, turbulence, flow gradients, and the like. Non-spherical particles that are elongated, e.g., particles that are generally oval or rod-shaped, can be distinguished from generally spherical particles by flickering in the scattered light as they rotate. Thus, the observable flickering will correlate with the flow properties, such as those listed above, e.g., by reduction in the flicker rate as viscosity increases. Thus, such elongated particles will provide additional characterization of flow properties in a system or device.
Preferably, but not necessarily, the detection of light scattering in the present methods is performed using simple instrumentation. By simple instrumentation is meant with magnification less than 500xc3x97, and without confocal imaging, and preferably without use of laser illumination. However, in some embodiments, laser illumination is advantageous, as laser may have important applications in providing incident light precision. Use of laser illumination can readily be used with sets of particles selected to provide multiple distinguishable characteristics, e.g., distinguishable intensities, distinguishing 2 or more different particles. Typically the sets of particles are selected by size and/or composition to provide the distinguishable characteristics. However, in some embodiments, it will be beneficial to use such apparatus, and/or to use electronic imaging devices. In addition, electronic image processing and analysis tools can also be used.
The term xe2x80x9cdynamic propertyxe2x80x9d refers to a property or characteristic of a system or material that changes over time. The property may, however, reach an endpoint. Examples include, without limitation, flow rate, mixing, distribution of a material, distribution of material during drying, and binding, distribution and/or number of function molecules or components, stability of material on a surface (e.g., as a function of experimental processing), presence of flow features such as turbulence or micro eddies or other extremely local flow dynamic effects. In particular embodiments, one or more of these dynamic properties is determined, at one or more timepoints or continuously over a time interval(s)
Detection may be performed using any of a variety of different detectors. One of ordinary skill in the art will be familiar with numerous detectors. For example, in some applications it may be sufficient for the particles to be viewed by eye. However, in other applications it may be preferable to use a film or electronic detector (e.g., a film or electronic camera), that produces a picture and/or electronic record. Such cameras may be still (which may be used in time-lapse manner) or video cameras. The film or electronic image can be further processed and/or analyzed to identify features (e.g., the number and/or position of particles) and/or to characterize one or more properties of such features. Of course, video cameras may be used with frame-grabbers to allow single or multiple image processing and/or analysis. Electronic cameras include, for example, charge coupled device (CCD) cameras, charge injection device (CID) cameras, and Complementary Metal Oxide Semiconductor (CMOS) cameras.
While the description of aspects and embodiments herein is generally presented in terms of fluids (used herein as equivalent to the term liquids), the analysis of flow behavior using RLS particles can also be performed in gaseous flow, e.g., air flow. Typically, but not necessarily, the velocities in such flow are greater than for liquids. Also typically, RLS particles used for gas flow analysis will be small, e.g., 1-40 nm, preferably 1-20 or 1-10 nm. In some applications it is also beneficial to use particles of lower density than solid metal particles. Examples include particles with a metal shell over a low density core, thereby maintaining high light scattering intensity but enhancing the ability of the particle to remain suspended in the gas for a useful period of time. The detection and analysis of particle distribution, flow pattern, and other properties in gas systems is essentially the same as for the fluidic systems described herein, and are part of the present invention.
Additional aspects and embodiments will be apparent from the following Description of the Preferred Embodiments and from the claims.